A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Knoll, Samantha; Ali, M. Yakut; Saif, M.T. A.

doi:10.3791/51873-v

Cited by 3 publications

(3 citation statements)

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“…Overall, the embedding of fiducial markers inside of the gels makes the accurate data analysis and the interpretation of traction force maps a challenging task. However, fluorescent beads can also be attached only to the gel surfaces, which improves control over their spatial distribution in the image plane and improves data quality 26 , see Fig. 1b.…”

Section: Resultsmentioning

confidence: 99%

“…To prepare PAA gels, we used a modified protocols of Refs. 10,26 . Briefly, glass bottom dishes (35 mm dish with 14 mm bottom well #1.5, Cellvis) were activated for 5 min with a solution of 3-(Trimethoxysilyl)propyl methacrylate (Sigma-Aldrich, 440159, diluted in absolute ethanol to the final concentration of 5%) and mixed with a final concentration of 3% diluted glacial acetic acid (diluted in water to the final concentration of 10%) to covalently link the polyacrylamide gels to the glass plates.…”

Section: Methodsmentioning

confidence: 99%

“…To prepare PAA gels, we used a modified protocols of Refs. 10,26 . Briefly, glass bottom dishes (35 mm dish with 14 mm bottom well #1.5, Cellvis) were activated for 5 min with a solution of Before imaging, the cells were washed twice with DMEM media and then switched to the imaging buffer, which includes DMEM media with the final concentration of 0.56 mg/mL of glucose oxidase (Aspergillus niger-Type VII, lyophilized powder, Sigma-Aldrich, G2133) and 34 μg/mL of catalase (bovine liver -lyophilized powder, Sigma-Aldrich, C40).…”

Section: Paa Gel Preparationmentioning

confidence: 99%

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Traction Force Microscopy with DNA FluoroCubes

Mortazavi,

Jiang,

Laric

et al. 2024

Preprint

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From cell differentiation to morphogenesis and cell migration, a multitude of processes are coordinated by mechanical forces that cells generate. Among diverse techniques to assess the mechanical properties of the cell, traction force microscopy (TFM) has emerged as one of the most popular methods for quantifying cell-generated stresses. Standard TFM procedures rely on fiducial markers in the extracellular environment to measure the deformations that are caused by cellular forces. Typically, fluorescent beads are used as fiducials. However, the replacement of beads with fluorescently labeled DNA structures can have numerous advantages, including a smaller size of the markers and the possibility of customizing the DNA structures, for example to read out orthogonal information or to realize a switchable surface functionalization. Here, we develop a multi-purpose platform for combining such setups with TFM. As fiducials we employ FluoroCubes - nanometer-sized DNA constructs - for TFM. These constructs are grafted to a high refractive index polyethylene siloxane surface for the precise tracking of displacements resulting from cell-generated forces. To ensure a local transmission of traction forces from the adhesion ligands to the substrate, we also graft RGD peptides, which represent the smallest ligands of the extracellular matrix, onto our elastic substrates. To further enhance the spatial resolution of the TFM, FluoroCubes can be supplemented with densely packed fluorescent beads as fiducials. We propose a modification of the Kanade-Lucas-Tomasi (KLT) optical flow tracking (OFT) algorithm for optimal, simultaneous tracking of FluoroCubes and beads. Together, the developed experimental setup and tracking algorithm yield highly resolved maps of traction forces that correlate well with the spatial distribution of kindlin at focal adhesions.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…To prepare PAA gels, we used a modified protocols of Refs. 10,26 . Briefly, glass bottom dishes (35 mm dish with 14 mm bottom well #1.5, Cellvis) were activated for 5 min with a solution of Before imaging, the cells were washed twice with DMEM media and then switched to the imaging buffer, which includes DMEM media with the final concentration of 0.56 mg/mL of glucose oxidase (Aspergillus niger-Type VII, lyophilized powder, Sigma-Aldrich, G2133) and 34 μg/mL of catalase (bovine liver -lyophilized powder, Sigma-Aldrich, C40).…”

Section: Paa Gel Preparationmentioning

confidence: 99%

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Traction Force Microscopy with DNA FluoroCubes

Mortazavi,

Jiang,

Laric

et al. 2024

Preprint

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Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts

Emon,

Joy,

Lalonde

et al. 2024

Acta Biomaterialia

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Trends in mechanobiology guided tissue engineering and tools to study cell-substrate interactions: a brief review

et al. 2023

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Sensing the mechanical properties of the substrates or the matrix by the cells and the tissues, the subsequent downstream responses at the cellular, nuclear and epigenetic levels and the outcomes are beginning to get unraveled more recently. There have been various instances where researchers have established the underlying connection between the cellular mechanosignalling pathways and cellular physiology, cellular differentiation, and also tissue pathology. It has been now accepted that mechanosignalling, alone or in combination with classical pathways, could play a significant role in fate determination, development, and organization of cells and tissues. Furthermore, as mechanobiology is gaining traction, so do the various techniques to ponder and gain insights into the still unraveled pathways. This review would briefly discuss some of the interesting works wherein it has been shown that specific alteration of the mechanical properties of the substrates would lead to fate determination of stem cells into various differentiated cells such as osteoblasts, adipocytes, tenocytes, cardiomyocytes, and neurons, and how these properties are being utilized for the development of organoids. This review would also cover various techniques that have been developed and employed to explore the effects of mechanosignalling, including imaging of mechanosensing proteins, atomic force microscopy (AFM), quartz crystal microbalance with dissipation measurements (QCMD), traction force microscopy (TFM), microdevice arrays, Spatio-temporal image analysis, optical tweezer force measurements, mechanoscanning ion conductance microscopy (mSICM), acoustofluidic interferometric device (AID) and so forth. This review would provide insights to the researchers who work on exploiting various mechanical properties of substrates to control the cellular and tissue functions for tissue engineering and regenerative applications, and also will shed light on the advancements of various techniques that could be utilized to unravel the unknown in the field of cellular mechanobiology. Graphical Abstract

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A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Cited by 3 publications

References 0 publications

Traction Force Microscopy with DNA FluoroCubes

Traction Force Microscopy with DNA FluoroCubes

Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts

Trends in mechanobiology guided tissue engineering and tools to study cell-substrate interactions: a brief review

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